Dry chemistry, lateral flow-reconstituted chromatographic enzyme-driven assays

ABSTRACT

A lateral flow chromatographic assay format for the performance of rapid enzyme-driven assays is described. A combination of components necessary to elicit a specific enzyme reaction, which are either absent from the intended sample or insufficiently present therein to permit completion of the desired reaction, are predeposited as substrate in dry form together with ingredients necessary to produce a desired color upon occurrence of the desired reaction. The strip is equipped with a sample pad placed ahead of the substrate deposit in the flowstream, to which liquid sample is applied. The sample flows from the sample pad into the substrate zone where it immediately reconstitutes the dried ingredients while also intimately mixing with them and reacting with them at the fluid front. The fluid front moves rapidly into the final “read zone” wherein the color developed is read against predetermined color standards for the desired reaction. Pretreatment pads for the sample, as needed, are placed in front of the sample pad in the flow path as appropriate. The assay in the format of the invention is faster and easier to perform than analogous wet chemistry assays.

This application is a continuation-in-part of U.S. application Ser. No.10/370,574 filed Feb. 24, 2003.

The present invention relates to conducting rapid, dry-chemistry,enzyme-driven chemistry assays using lateral flow chromatography.

BACKGROUND OF THIS INVENTION

The human enzyme glucose-6-phosphate dehydrogenase (“G6PD”) performs acritical function in human biochemistry. It is part of the oxidativepentose pathway, wherein it functions to minimize oxidative attacks offree radicals upon cells by providing reducing equivalents—i.e., G6PDconverts glucose-6-phosphate to 6-phosphoglutonate, thereby liberating aproton that reduces nicotinamide adenine dinucleotide phosphate, NAPD,to NAPDH. The NAPDH initiates a series of downstream reactions thatultimately reduce the free radical oxidizing agents and render many ofthem ineffective in normal human biochemistry.

G6PD is present in all human cells, but it is in higher concentration inred blood cells which, in one of their primary functions, act as oxygentransport vehicles and are hence particularly susceptible to oxidativeattack. The efficiency of the G6PD system is remarkably high asreflected by the fact that during normal activity less than 1% of itscapacity is utilized in combating and preventing undesirable oxidativeeffects. However, when strong oxidizing agents, such as members of thequinine class of anti-malarial drugs must be introduced to humans, theneed for rapid production of reducing agents is greatly increased.

Several mutations of the gene which encodes for G6PD are known whichdecrease the efficiency of the enzymes in the biochemistry ofindividuals processing such a mutation in both halves of their genome,causing the quantity of their G6PD to remain at the same level as inpeople with a normal gene, but also causing their G6PD to show greatlyreduced specific activity. In these individuals, administration ofstrong oxidizing agents such as members of the class of quinine-typeanti-malarials, may cause severe clinical complications, such ashemolytic anemia, because the low specific activity of their G6DP doesnot enable the production of sufficient reducing agents to prevent rapidunwanted oxidative effects on their red blood cells. In areas wheremalarial infections are common and at times even epidemic, a needtherefore exists for a rapid efficient test that will readilydistinguish persons having G6PD of low specific activity from personswhose G6PD activity is normal and will enable medical personnel toensure that (1) the quinine antimalarials are prescribed only forindividuals with normal or better G6PD specific activity and (2) personswith lower than normal G6PD activity are medicated with an alternativetype of anti-malarial drugs.

Heretofore, assays that involve enzyme activity, in any context, havemost usually been conducted in “wet chemistry” formats which requiretrained laboratory personnel to prepare for and perform them. Thereagents for such assays must either be made fresh from dry componentsor be reconstituted from commercially available dried formulations. Wetreagents are less stable than dried ingredients or dried formulationsand, to the extent they must be stored, more stringent, carefullymonitored storage conditions, including special handling techniques toprevent contamination, are required. Those assays also requireinstruments such as spectrophotometers, fluorimeters or other suchinstrumental equipment to read the endpoint results of the assay. Suchassays are not practical for use in doctor's offices, hospitals andnursing home facilities, under epidemic conditions, or for home or fielduse.

Automated clinical chemistry analyzer systems are in industrial usewhich perform dry chemistry formatted assays wherein the presence,absence, concentration or specific activity of a substance present in orabsent from a sample is determined. Such a substance is, for purpose ofthis application, referred to as the “analyte” and it may be an enzymeper se (as in the G6PD assay hereinafter described in detail) or asubstance necessary to the elicitation of a specific enzyme activity.Examples of automated clinical chemistry analyzer systems are theJohnson and Johnson Vitros™ and the Roche Cobas™ systems. These andsimilar automated systems are not subject, when performing as designed,to the preparation skill requirements and shelf life problems associatedwith humanly performed dry chemistry assay work. Because programmedrobots perform the manipulative tasks, the need for intensively trainedhumans is likewise avoided. The systems, however, require on boardreader instrumentation and they are necessarily too large, toocomplicated and generally too burdened with infrastructure requirementsto be practical for use in doctor's offices or homes and in manyhospitals, clinics and like places. Clearly, they have too manytechnical requirements for field use.

There are available, as well, a very few non-instrument baseddry-chemistry assays, such as the Orasure QED™ assay for alcohol whichis based upon use of the alcohol dehydrogenase enzyme to determinealcohol content of saliva in the field. This and other known assays ofthis genre have heretofore been limited to determinations that can bemade on samples that are free of substances that may obscure, inhibit orin some other manner intrinsically interfere with and render imprecisedeterminations that are dependent upon some aspect of enzymatic actionor content.

An example of an enzymatic assay that operates on samples containingvisually obscuring substances and uses antibody capture zones to selectfor enzyme analytes is shown in U.S. Pat. No. 5,506,114. This systemrequires wash steps to remove the visually obscuring substances and issufficiently cumbersome to perform that it is impractical for field useor use in doctor's offices, homes, most clinics and many hospitals andthe like.

BRIEF DESCRIPTION OF THE INVENTION

In its broadest aspect, this invention rests upon the recognition thatrapid, dry chemistry, enzyme-driven assays may advantageously beconducted using lateral flow chromatography, wherein predeposited drysubstrate, as hereinafter defined, is reconstituted chromatographicallyby the lateral flow of liquid sample and entrained substrate through atleast one region of a lateral flow device, with production of acolorimetric reaction at the forward flow front of the sample-substratemixture in the endpoint or “read” zone of the chromatographic device.The color produced is that typical of the endpoint color of thecorresponding wet chemistry clinical assay, obtained in the region ofthe device where forward flow ceases, i.e., the zone that is farthestfrom the point of sample introduction. It is within the scope of theinvention, depending upon the specific assay being conducted, to includechromatographic regions in the device that remove interfering substancespresent in the sample and/or regions that have been treated topreconcentrate the analyte before it moves into the endpoint reactionzone. In some assays, at least one substance heretofore deemed tointerfere with the endpoint observation, i.e., hemoglobin, need not beremoved since its otherwise endpoint-obscuring color deposits equally inthe endpoint zone and the zone just preceding it. The result in thiscase is that the endpoint is easily observable by direct comparison ofthe color produced in the endpoint zone with that of the unreacted redcolor in the abutting, immediately preceding zone.

In general, in the lateral chromatography, enzyme-driven assays of thisinvention, the movable, predeposited dry subtrate is placed near to andjust beyond the junction of the sample receiving pad and the next pad inthe sample flow path on the chromatographic strip. It may, however beplaced elsewhere in the sample flow path to accommodate particularrequirements of either the sample or one or more ingredients in thesubtrate, so long as it is placed in the flow path substantially beforethe endpoint, or “read”, zone where sample flow stops and any excessfluid present runs off into an absorption pad or other sink device thatmay be provided.

It is important that the dried substrate be deposited within a tightlyconfined area so as to facilitate its being completely picked up by theforward flow of the sample. The placement in the flow path of the driedsubtrate should also take into consideration that reconstitution of thesubtrate in dissolved or dispersed form within the liquid sample isdesirably completed by the time the sample reaches the point wheresample flow ceases.

It has also been found that the present enzyme-driven lateral flow assaycan be combined with known solid phase isolation methods, and that in atleast some instances when this is done, the sensitivity of detection ofthe desired end point is enhanced substantially.

The format of such methodology involves binding a ligand for the targetenzyme to a particulate solid support material—e.g., disks of filterpaper or other common solid support material, such as but not limited tonitrocellulose, nylon, polyethylene, etc. or superparamagnetic particlesand the like—by coupling, coating, impregnation or any other knownmethod. The ligand-bound particles are then mixed with a sample of fluidknown to contain the target enzyme and incubated for a period requisiteto allow binding of target enzyme to the ligand. The particlescontaining enzyme-ligand reaction products bound thereto are thenseparated from the fluid sample by known separation techniques,including filtration, sedimentation, centrifugation and in the case ofsuperparamagnetic particles, subjection to the influence of a gradientmagnetic field of sufficient strength.

The collected particles containing bound enzyme-ligand reaction product,after separation from the initial sample, are then suspended in a volumeof known buffer selected as one known to be appropriate to theenzyme-ligand reaction product and this buffer suspension is utilized asthe sample in an enzyme-driven test assay constructed for determinationof enzyme concentration or some other parameter of the enzyme. Thisapproach is of special value in a method for beta-lactamase enrichmentand detection, wherein all beta-lactamase present in a bacterial cultureor in sample of human fluid, such as a nasal wash, or a urine sample, ispreconcentrated by immunological separation via ligand-bound particlesand is then transferred into the uptake volume of the enzyme-driventest.

For convenience of shipping, storage and use, each chromatographic stripof this invention is preferably housed within a suitable deviceconstructed so that the strip is positioned laterally. Many such devicesare well-known in the art and any of them constructed so that theperformance of an assay on the chromatographic strip positioned withinit is performed by lateral flow may appropriately be utilized.

This format for conducting enzyme-driven assays has a number ofadvantages as hereinafter described in detail.

A specific G6PD assay that can easily be used successfully by anyoneoperating in the field, the home, a doctor's office or at any site wheretrained laboratory personnel and instrumentation are lacking, isspecifically described hereinafter and depicted in the accompanyingdrawings, as are assays for total serum cholesterol, beta-lactamaseactivity, and peroxidase activity.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1A represents a chromatographic strip preprepared for theperformance of the G6PD assay of this invention.

FIG. 1B shows the same strip after the sample has been applied to it andbefore the sample has reached the endpoint zone.

FIG. 1C shows the strip as it appears when the assay is completed.

FIG. 2A is a chart showing data obtained from use of the G6PD test ofthis invention.

FIG. 2B is a graph of time in seconds elapsed from the cessation offorward flow at the end of the strip to the appearance of purplish bluecolor in the endpoint zone, plotted against G6PD activity level for thesamples, measured activity level of which is shown in the FIG. 2A chart.

FIG. 3A is a chart showing data obtained in the test of this inventionfor total serum cholesterol.

FIG. 3B A plot of time in seconds elapsed from cessation of forward flowat the end of the strip to appearance of blue/purple color in the “read”or endpoint zone, versus total serum cholesterol in mg./deciliter.

FIG. 4A is a chart showing data obtained with whole blood in the totalserum cholesterol test, as described in Example 2 hereof.

FIG. 4B is a plot of time in seconds to appearance of bluish purplecolor against cholesterol in mg. per dl.

FIG. 5A is a chart of data obtained using strips of this invention inmeasuring β-lactamase activity, using commercially available β-lactamasestandards.

FIG. 5B is a plot of time in seconds to appearance of bluish purplecolor against standard activity in U/ml, where “U” represents units ofactivitiy.

FIG. 6A is a chart obtained from measuring β-lactamase activity in aculture of E. coli known to produce β-lactamase.

FIG. 6B is a graph of the data from FIG. 6A showing time in seconds toappearance of bluish purple color against β-lactamase activity measuredin CFU (colony-forming units) per ml.

FIG. 7 is a chart of the data from the measurement of peroxidase asdescribed in Example 4, infra.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of describing this invention in its broadest scope, thefollowing four definitions apply wherever the terms appear in thisapplication:

-   -   (1). “Sample” refers to any liquid biological or environmental        matrix or any liquid extract or liquid concentrate thereof that        is to be assayed.    -   (2). “Analyte” refers to a target substance of the assay which        may be present in, or absent from, the sample. The analyte may        itself be an enzyme or it may be a substance required to elicit        specific enzyme activities, such as substrate, co-substrate or        cofactor.    -   (3). “Substrate” refers to a combination of those components        necessary to elicit a specific enzyme reaction, which components        are not present in the sample or are present therein in        insufficient quantity where the analyte is not an enzyme, or        where the initial enzyme reaction drives a cascade of additional        reactions requisite to the desired final determination. The        substrate may contain any combination of cofactors, substrates        as defined in the preceding sentence, co-substrates, dye or        colorimetric components, or enzymes themselves.    -   (4). The term “dry-chemistry” refers to an assay format where        the components required for a given determination on a sample        are maintained in dry form until reconstituted by the        performance of the assay itself, rather than being reconstituted        prior to and separate from the assay procedures.

Reconstituting substrate components necessary in enzyme-driven assays bylateral flow chromatography exhibits a host of advantages in comparisonto methods that do not utilize such chromatography. At least some ofthem are identified below:

In the usual manual performance of enzyme driven assays, it is necessaryto dilute the sample serially in order to control rapid enzyme kinetics;in the lateral flow assays of this invention, sample dilution isunnecessary because only a very small sample is applied to thechromatographic strip upon which the assay is performed.

The sample itself reconstitutes the substrate, thereby eliminating theneed for separate reconstitution buffers and steps.

This chromatographic reconstitution of the subtrate increases thesubstrate concentration available to the sample over that provided inthe usual laboratory performance of a comparable assay.

The chromatographic media utilized are any of those well-known andwell-characterized in the art. Their known characteristics may readilybe taken advantage of, in particular assays where this is desirable, byreserving a zone of the strip in which to concentrate analyte byremoving some of the fluid present therein or by reserving a zone of thestrip and pretreating it appropriately so that substances present in thesample that tend to inhibit or interfere with the desired enzymereaction are wholly or partially immobilized in that zone, or theirflowability is retarded there.

Also, inasmuch as the endpoint color formation is restricted to a singleendpoint zone, it is much easier to read and evaluate, even withoutinstruments such a spectrophotometers, than when color is diffusedthroughout a large liquid volume.

Further, the action of the sample in picking up dried substrate at itsfluid front results in leaving an essentially substrate free zoneimmediately adjacent to the endpoint zone. As the sample moves forward,there is clear differentiation between any residual color fromhemoglobin and the desired endpoint color, forming at the fluid front.This eliminates any need, in some cases, to remove any substance in thesample (such as the red of hemoglobin) that is known to produce visuallyobscuring color when manual chemistry tests with liquid samples areperformed.

Still further, the relative speed from sample introduction to endpointreaction that characterizes chromatographic lateral flow tests providesgreat advantages in the enzyme driven tests. These tests, when conductedby classical manual analytical methods often require digestion times forcontact between sample and substrate that are in the order of 30-45minutes and even longer. Notably, these times do not include the timeneeded in these classical manual methods for making dilutions,conducting concentration steps or substance removal steps, and the likemanipulations. By contrast, the lateral flow assays can usually beconducted in time periods within a 5-20 minute range, starting from theintroduction of the sample to the strip and ending with evaluation ofthe endpoint result.

To measure an analyte in red blood cells, such as the G6PD analyte ofExample 1 herein, the red blood cells must be lysed (split open) withsurfactants or other lysing agents before the analyte can be measured.Another such analyte normally found in red blood cells, an assay formeasuring which in the dry chemistry format of this invention canreadily be devised, is pyruvate kinase.

There are also many instances where the analyte is normally present inblood serum or plasma, rather than red blood cells, that areenzyme-driven and may beneficially be converted to the “dry chemistry”format of this invention. Among them are such tests as those forglucose, cholesterol, HDL-cholesterol, triglycerides, urea nitrogen,creatinine, alanine aminotransferase (ALT), aspartate aminotransferase(AST), lactate dehydrogenase (LDH), creatinine kinase (CK) and the like.

The present invention will likewise be useful in instances where theanalyte is present in other biological fluids, such as measuring alcoholin saliva or measuring drugs such as acetaminophen or salicylate inurine.

Overall, the present invention is perceived as having special utility insituations where:

-   -   (A). the typical concentration of analyte in the sample is very        low such as in the creatinine and uric acid test for measuring        kidney function; where the intimate mixture of sample and        substrate at the fluid front will improve the sensitivity of the        test compared to that of classical manual methods;    -   (B). screen testing providing a qualitative “yes” or “no” result        is conducted to determine the onset or severity of a disease,        such as the ALT (alanine aminotransferase) and AST (aspartate        aminotransferase) screens for liver damage, especially on        patients who are taking drugs capable of causing liver damage,        and screens conducted on infants for genetically caused        disorders, such as the test for phenylketonuria (PKU) or the        test for galactose used to detect galactosemia; and the like;        and    -   (C). tests where some portion of a sample needs to be removed        prior to measurement of another portion—e.g. the measurement of        HDL (high density lipids)-cholesterol, wherein LDL (low density        lipids) and VLDL (very low density lipids) need to be removed,        by precipitation or otherwise, before total HDL-cholesterol is        measured.

As is apparent from the foregoing, the invention is adaptable to bothqualitative and quantitative formats. Some other milieus, in addition tothose mentioned above, in which it is amenable to being widely appliedare those wherein enzyme-labeled antibodies have been used in wetchemistry methods, to detect the presence of a suspected antigen in anunknown sample, followed by reacting enzyme-tagged antibody-antigenreaction product with an appropriate color-producing agent. These testshave been traditionally performed both qualitatively and quantitativelyin wet chemistry formats. Adapting them to being performed according tothis invention will produce all the benefits of speed, ease ofmanipulation, and the like that are noted hereinfore other enzyme-drivenreactions.

Particular attention has been given to date to the application of thisinvention to whole blood chemistry tests.

For example, glucose may be determined by a reductive assay oftenutilized in blood taken from diabetic patients. To perform this assayaccording to the present invention, the immunochromatographic strip isprepared with predeposited dry ingredients consisting of glucosedehydrogenase, nicotinamide adenine dinucleotide (“NAD”), nitro bluetetrazolium and diaphorase. Glucose reduces to its hydride, and thehydride in the presence of diaphorase reduces nitro blue tetrazolium toprovide purplish blue color in the “read” zone.

In the oxidative assay for glucose the dry ingredients deposited in thesubstrate zone are glucose oxidase, peroxidase, 4-aminoantipyrine andphenol, or a derivative of phenol. Glucose and glucose oxidase in thepresence of oxygen produce hydrogen peroxide, which in turn reacts withaminopyrine and phenol (or a phenol derivative) to produce a distinctcolor in the “read” zone.

A cholesterol assay often utilized in performing a blood lipid profileis conducted according to this invention by depositing cholesterolesterase, cholesterol oxidase, 4-amino-antipyrine and a phenolderivative, all in dry form, in the substrate zone. When a blood sampleis introduced to the strip, lysed and allowed to flow along the strip, adistinct color is observed in the read zone that has an intensityproportional to the cholesterol concentration in the sample. Thispermits the development of color standards by well known methods. Suchstandards are conveniently used in situations where instruments forreading color intensity are not readily available, e.g doctor's offices,the home, in the field, etc.

A test according to this invention for measuring HDL-cholesterol employsa preprepared ICT strip wherein a first zone immediately following the“lyse” zone is provided with deposited, immobilized precipitatingreagents that capture and bind the low density (“LDL”) and very lowdensity lipids (“VLDL”) in the sample, allowing only the HDL-cholesterolin the sample to pass into the substrate zone. The ingredients in thesubstrate zone are again dry cholesterol esterase, cholesterol oxidase,4-aminoantipyrine and a phenol derivative and the color produced in the“read” zone is proportional in intensity to the concentration ofHDL-cholesterol in the sample.

To perform the creatinine assay for kidney malfunction assessment by themethod of this invention, the predeposited dry ingredients in thesubstrate zone are creatinine immunohydrolase, sarcosine oxidase,peroxidase, 4-aminoantipyrine and a phenol derivative.

An ALT assay for liver malfunction can be performed according to thisinvention on an ICT strip wherein the dry ingredients predeposited inthe substrate zone are pyruvate oxidase, peroxidase, 4-aminoantipyrineand a phenol derivative.

Preparing an ICT strip as herein described with predeposited dryingredients in the substrate zone consisting of creatinine, adenosinetriphosphate, phosphophenol pyruvate, pyruvate oxidase, peroxidase,4-aminoantipyrine and a phenol derivative enables performance accordingto this invention of an assay for congestive heart failure.

Where phenylketonuria (PKU) is suspected in neonates, this inventionprovides a convenient “yes-no” assay wherein an ICT strip is equippedwith a dry deposit of phenylalanine dehydrogenase, nicotinamide adeninedinucleotide and diaphorase. When a blood sample is added and colorappears in the read zone, the test is positive for PKU; if no colorappears, the disease is not present.

Tests for alcohol content in blood can be run both oxidatively andreductively in the method of this invention. A test strip for thereductive test can be prepared by depositing alcohol dehydrogenase,nicotinamide adenine dinucleotide, nitro bluetetrazolium and diaphorasein dry form in the substrate zone; an oxidative test for the samepurpose employs an ICT strip in the substrate zone of which ispredeposited dry alcohol oxidase, peroxidase, 4-aminoantipyrine and aphenol derivative. In either case, the color in the endpoint, or “read”zone will be proportional to the alcohol content of the blood sample.

An assay for acetaminophen (Tylenol R) in blood can conveniently be runaccording to this invention. The ICT strip for this purpose will containpredeposited dry aryacylaminidase, ortho-cresol, and an oxidizing agent,such as sodium bisulfate. When a blood sample is added one can determinewhether or not the patient has overdosed on the acetaminophen bycomparing its color intensity to that of a preestablished colorstandard.

A rapid dry-chemistry lateral flow assay was devised to illustrate thisinvention specifically. The detection of G6PD deficiency was selectedfor this purpose because of the perceived need for a reliable test forthis purpose that can be conducted in the field, without instrumentationand in the absence of trained laboratory personnel. Assays for totalserum cholesterol, beta lactomase activity and peroxidase activity arealso exemplified.

SPECIFIC EXAMPLES Example 1

This test is performed on a lateral flow strip as pictured in FIG. 1A,having a “lyse” or, wicking, pad from which the sample flows forwardinto the second, or substrate pad. The latter pad has two regions. Thefirst such region is a tightly confined substrate zone in which ismovably pre-deposited all of the dried components that may beconventionally used in the art to enable G6PD in the sample to reducethe faint yellow dye nitroblue tetrazolium, to dark blue formazan. Therate of conversion of nitroblue tetrazolium to dark blue formazan is oneof several “wet chemistry” tests that has been used in the art tomeasure G6PD specific activity. The substrate pad also contains what isinitially a substrate-free zone, positioned at the farthest end of thestrip from the sample introduction point. As the sample picks upsubstrate and flows forward the initially substrate-free zone becomesthe “read” or endpoint zone when the sample containing reconstitutedsubstrate occupies it and forward flow ceases. Prior to the endpointzone, as the sample moves along the strip its forward flow momentumpicks up the dried substrate components and, as it moves to the end ofthe strip, reconstitution of the picked up dried ingredientsconcentrates in the fluid front, which rapidly becomes the endpoint or“read” zone where color develops. The area of the strip just prior tothe read zone from which dried substrate has been removed by the fluidfront then becomes essentially substrate free, but meanwhile has beencolored red by the hemoglobin in the sample that passed through thezone.

A specific advantage of the lateral flow chromatography format for thistest over a “wet chemistry” test method is realized because the sampleof choice for G6PD activity determinations is blood. This is because, asearlier noted, blood cells contain the vast majority of each humanindividual's G6PD. The blood cells must be lysed to make the enzymewhich codes for G6PD available for the reaction. Lysis i.e. (splitting)of blood cells releases hemoglobin and imparts a red color to the samplewhich must be removed or at the least, substantially diminished inintensity, when the test is conducted by wet chemistry methods becausethe blue of formazan is extremely difficult, verging on impossible, todiscern visually in a diffuse liquid sample to which a uniform red colorhas already been imparted. The blue color of formazan in such a diffuseliquid sample can readily be seen, as it emerges, as a mere darkening ofthe initial red color; moreover, different individuals attempting to seethe color change typically interpret any given test differently. In thechromatographic test herein described, however, the red color need notbe removed because the reaction of sample and chromatographicallyreconstituted dried ingredients occurs in a well defined flow region atthe fluid front, so that when flow terminates at the end of thechromatographic strip and excess fluid, if any, in the sample runs intoa sink, two adjacent regions of the strip are clearly visible. The oneclosest to the end of the strip, the endpoint or “read” region, is apurplish blue color, while the adjacent region exhibits the red color ofhemoglobin and the two are clearly discernable from one another.

In the actual tests performed with the G6PD test device, a human wholeblood sample was drawn into a tube containing heparin to preventclotting of the sample. A portion of the sample was first assayed forG6PD activity using a clinical “wet chemistry” laboratory procedure ofthe prior art and, by ultraviolet spectrophotometric analysis wasconfirmed to have a normal human G6PD activity level of 116 U/di. Aportion of the sample was lysed by adding aqueous 10% Triton X-100 in avolume ratio of blood to lysing solution of 10:1. The resulting 10%Triton X-100, 90% whole blood lysate was incubated at 37° C. for 6 hoursto allow proteolytic degradation of its initial G6PD activity. Using thesame conventional “wet chemistry” laboratory procedure as used on aportion of the whole blood, this sample was found to have no G6PDactivity.

A second lysate sample was produced in the same manner as the first.Dilutions of the fresh lysate and the degraded lysate were produced withtargeted G6PD activities of 104,80,40 and 20 U/dl. The actual activityof each of these samples was measured using the same conventional “wetchemistry” lab procedure. The results of the fresh lysate samples wereconsistent with the targets established for them, while the degradedsamples consistently showed no activity.

To the sample receiving end of each of a series of separately preparedchromatographic G6PD strips mounted laterally on a support there wasadded 45 μl of each of the fresh and degraded lysate samples. Thesamples were allowed to flow chromatographically along the laterallyplaced strips to their terminal ends. As each sample reached theterminal end of a strip, timing was initiated and the time needed forthe visible purplish blue color to appear in the endpoint zone wasrecorded. The activity levels of the fresh lysate samples are shown inthe chart that is FIG. 2A. The times to purplish blue color appearanceare graphed against enzyme activity level for each fresh lysate samplein FIG. 2B. All concentration levels of the fresh lysate had G6PDactivity and eventually produced the purplish blue color in the endpointor “read” zone of the strip as expected.

These experiments were necessarily performed with pre-lysed bloodsamples because no G6PD deficient blood samples were available and itwas necessary to test a range of G6PD levels to validate the test. Lysisof blood samples at the sample introduction end of a chromatographicstrip is a procedure known in the art and is intended to be performed inthe known manner on these test strips in actual practice with samples ofunknown G6PD activity, thus obviating the need for any pre-test sampletreatment. The tests described above were considered necessary for thepurpose of establishing a timed endpoint so as to enable users of thetest strips in the field to distinguish readily between G6PD deficientblood and G6PD normal blood.

The clinically relevant G6PD deficiency level has heretofore been fixedat 20 U/dl. The assay is designed to be run at ambient temperatures ashigh as 37° C., which typically occur in warm climates where malariainfections are prevalent and the need to identify G6PD-deficientindividuals before prescribing malaria medication is acute. The rate ofG6PD enzyme activity at 37° C. is known to be double that at normal roomtemperature of 25° C. For conducting the G6PD chromatographic test, atarget activity level cutoff was set at 50 U/dl at room temperature(corresponding to 25 U/dl at 37° C.). By setting the test endpoint at 70seconds from the time the sample and entrained reconstituted substratereach the terminal end of the strip, discrimination between individualswith normal G6PD levels and those whose G6PD levels are clinicallydeficient can readily be made in the field.

In the regions of the world where malaria is most prevalent, G6PDdeficiency is relatively common. The ease of performance of the test bynon-laboratory trained personnel and its speed combine to indicate thatthe use of the lateral flow chromatography G6PD test of this inventionwill have substantial value in ensuring that malaria-infectedG6PD-deficient individuals no longer receive medications for malariathat materially exacerbate their health problems.

Example 2

As previously noted, the measurement of cholesterol concentration in aliquid sample can be performed by the enzyme-driven chromatographicassay of this invention in a manner similar to that for G6PD. A lateralflow test strip was constructed by depositing all of the reagentsconventionally used in wet chemistry tests for total serum cholesterolplus a color-producing ingredient required to produce a color change ata rate proportional to the concentration of cholesterol constituents.The phenol derivative used in this experiment was TOOS i.e.,3-(N-ethyl-3-methylanilino)-2-hydroxypropane sulfonic acid (Sigma,E-8631) which produces a blue/purple color that can be readilydistinguished above the hemoglobin background of a whole blood sample. Acommercially available panel of serum cholesterol standards (Sigma,C-0534) was run on these test strips to confirm that the time tovisualization was dependent on cholesterol concentration. A clear dosedependence was observed as seen in FIG. 3A and its plot, FIG. 3B.

To evaluate these test total cholesterol strips with a whole bloodsample, a panel of whole blood samples was constructed. Rabbit blood wasused to simulate extreme hypocholesterolemia because the rabbit nativeserum cholesterol level is very low, in this case 38 mg/dl. For thebalance of the tests at higher levels, this blood was centrifuged andthe serum fraction was withdrawn and separated into aliquots. To eachaliquot was added an equal volume of each of the serum cholesterolstandards referred to above.

The resulting panel of adjusted whole blood samples was assayed with acommercially available wet chemistry spectrophotometric assay (ThermoTrace, cholesterol concentration from 38 to 328 mg/dl). Each of theseadjusted samples was lysed and assayed on a test strip as describedabove. Again, a clear dose dependence was observed in these whole bloodlysate samples. This dependent relationship is apparent from FIG. 4A andthe corresponding data plot, FIG. 4B.

In each of FIGS. 3B and 4B, y=cholesterol in mg. per dl. and x=time inseconds to visualization of color in the read zone. The symbol R² is thecorrelation coefficient of the data to the curve drawn.

Example 3

The beta-lactams are a class of antibiotics, including penicillins andcephalosporins, which contain a characteristic beta-lactam ringstructure. This structure interferes with enzymes needed for thesynthesis of peptidoglycan to produce defective cell walls in dividingbacteria and renders these walls susceptible to lysis by osmoticpressure. One mechanism of bacterial resistance to these antibiotics isthe production of beta-lactamase enzymes, which specifically cleave thebeta lactam ring, rendering the antibiotic ineffective and restoring theability of the bacteria to multiply successfully.

A lateral flow enzyme-driven chromatographic test strip to detect thepresence of β-lactamase was constructed by depositing all of thereagents required according to prescribed prior art wet chemistrymethodology, to produce a color change at a rate proportional to theconcentration of β-lactamase. As in Example 1, the liquid samplechromatographically reconstitutes the dry ingredients deposited on thechromatographic strip that measure beta lactamase activity, in thisexample a chromagnic cephalosporin (Oxoid, Sr0112C). Color formation isobserved in the reaction zone at the distal end of the strip only ifβ-lactamase was present in the original sample volume.

To confirm that the time to visualization of color with these strips wasdependent on β-lactamase activity, a commercially available purifiedβ-lactamase standard (Sigma, P-4524) was obtained, reconstituted,diluted to a range of β-lactamase activities and run on these teststrips. A clear dose dependence was observed as shown in FIG. 5 A andits data plot, FIG. 5B.

A sample of E. coli bacteria known to produce β-lactamase (ATCC #35218)was obtained and grown by culturing according to the directionsaccompanying it. The presence of β-lactamase activity in the culturedbacteria was confirmed by a conventional wet chemistryspectrophotometric assay. Next the concentrated culture was diluted, itscell concentration was calculated from a turbidity measurement and aseries of dilutions were run on the same test strips for beta-lactamaseactivity referred to earlier in this example. Here again, a clear dosedependence was observed in these diluted bacterial culture samples. Thisdependent relationship is apparent from FIG. 6A and its plot, FIG. 6B.In FIGS. 5B and 6B, y represents units of beta-lactamase activity and“Ln” is the natural logarithm of the number “x”, which represents themeasured time in seconds to visualization of color in the endpoint orread zone. In FIG. 6B, the symbol “E” stands for a factor of 10, while“E” followed by a plus sign and a number signifies 10 to the positivepower of the number—e.g., 1.00 E+08 means 1×10⁸ “E” followed by a minussign and a number means 10 to the negative power of the number—thus inFIG. 6B where “R²=9.955E-01”; “E-01”=10⁻¹ and R² is 0.9955. In bothFIGS. 5B and 6B, R² is the correlation coefficient of the data to thecurve drawn.

Current methods of detecting beta lactamase enzymes require that thebacteria suspected of having developed antibiotic immunity be culturedto expand the size of the bacterial colony and consequently expand theamount of β-lactamase present. The colony is then incubated with disksthat have been preimpregnated with chromogenic cephalosporins. Each diskabsorbs a very small volume of the sample, and any β-lactamase enzymepresent cleaves the chromogenic cephalosporin to produce a pronouncedcolor change in the disk.

In an effort to improve accuracy, efficiency and sensitivity of themethod, superparamagnetic particles of sufficient magnetic moment to beseparated by exposure to the magnetic field of a rate earth magnet areconjugated to antibodies specific to the β-lactamase enzyme. Theseparticles are then added to a large volume of sample from an infectedpatient whose response to antibiotics is unsatisfactory. In the case ofresistant upper respiratory disease, for example, the sample is a nasalwash. When the suspect bacteria are urinary tract pathogens, a urinesample is ideal. The sample and the particles are incubated long enoughfor reaction to occur. The magnetic particles bearing theenzyme-antibody conjugates are separated by the magnetic field of therare earth magnet from unreacted particles and the liquid sample. Theseparticles are then released from the magnet and, resuspended in a smallvolume of buffer compatible with the test reagents on thechromatographic test strip for β-lactamase. Preliminary evaluations ofthe current practice with chromogenic cephalosporin-impregnated discsand enlarged bacterial colonies obtained by culturing the pathogens froma patient against the efficiencies expected to be realized by thetechnique described strongly suggest that markedly improved sensitivitywill be realized. Morever, obtaining the information rapidly may in manycases save a patient's life by indicating the need for a quick switch tomedication containing no β-lactam.

Example 4

Recent literature, including an article in the New England Journal ofMedicine (Prognostic Value of Myeloperoxidase in Patients with ChestPains: Vol. 349#17, 1595-1604), highlights the potential importance ofMyeloperoxidase as a marker of cardiac disease. As a potentialdiagnostic tool, a prototype enzyme-driven chromatographic assay wasconstructed to detect peroxidase activity in either serum or wholeblood. A lateral flow test strip was constructed by depositing all ofthe reagents required in existing wet chemistry methodology to produce acolor change at a rate proportional to the peroxidase activity of a testsample.

Briefly, glucose oxidase (Sigma, G-2133), 4-aminoantipyrridine (Sigma,4382) and the phenol derivative TOOS (Sigma, E-8631) were combined insolution, applied to the sample receiving end of a chromatographic stripand dried. Similarly, a solution of glucose (Sigma, G-7528) wasprepared, applied to the distal end of the same strip in a volume suchthat an empty void space remained between the treated regions and nomixing occurred, and dried. Upon reconstitution of these driedcomponents, as is known in the art, the glucose and glucose oxidaseproduce hydrogen peroxide which can be utilized by any peroxidaseactivity in the sample to produce a characteristic purple color from thepreviously colorless phenol compound.

As hemoglobin, which may be present in small quantities in a serumfraction of whole blood and will be present in large quantities inunseparated whole blood has intrinsic pseudoperoxidase activity,additional reagents were included to insure this activity did notinterfere with the tests. Sodium nitrite is a strong oxidizing agentwhich will rapidly oxidize hemoglobin and eliminate its pseudoperoxidaseactivity as noted in U.S. Pat. No. 6,200,773. Sodium nitrate (Sigma,S-2252) was added to the first reagent zone of the above described teststrip in sufficient quantity to eliminate the pseudoperoxidase activityof a moderately hemolized human serum sample.

To determine whether the time to visualization with these strips wasdependent on the peroxidase activity in the test article, a commerciallyavailable horseradish peroxidase standard (Sigma, P-8375) was obtainedand reconstituted. This standard was then diluted to a range ofperoxidase activities in moderately hemolized human serum and run onthese test strips. A clear dose dependence in time to visualization wasobserved as reflected in FIG. 7.

Using the dry chemistry, lateral flow-chromatography format describedherein, most enzyme driven tests can readily be rendered easier, fasterand cheaper to perform. Combining chromatographic separation techniquessuch as ion exchange, specific affinity, size exclusion and the likewith the dry chemistry, lateral flow test format will in many instancesproduce even greater benefits.

Those skilled in the art of designing lateral flow chromatography testswill readily perceive ways of applying these techniques to transformmuch of what is now performed as clinical wet chemistry into low cost,convenient tests performable in virtually any location by almost anyone.In particular, various tests are well known to be performable withalternative dry reagents to those specifically mentioned herein for usethe substrate zone. Likewise, the ICT strip may be prepared to enablethe performance of additional steps prior to the contact of sample withthe dry ingredients in the substrate zone, without departing in any wayfrom the spirit of this invention. It is therefore intended that thepresent invention be limited only by the appended claims.

1-19. (canceled)
 20. An assay device comprising at least two pads influid communication and positioned on an adhesive backing, wherein (1)the first pad is a sample receiving pad adapted to receive a samplewhich flows through the sample receiving pad, and (2) the second pad isa substrate pad on which has been deposited a mobilizable dry mixture ofcomponents including a substrate for an enzyme, which components, whenreconstituted by forward flow of the sample, perform an assay on thesample.
 21. The assay device of claim 20, wherein a result of the assayperformed on the sample is determined by a color indicative of thepresence or concentration of the enzyme in the sample.
 22. The assaydevice of claim 20, wherein a region of the first pad has been treatedto remove or reduce the concentration of a substance in the sample asthe sample moves through the first pad, which substance is known tointerfere with, obscure the result of or otherwise hinder theperformance of the assay.
 23. The assay device of claim 20, wherein aregion of the first pad has been treated to concentrate the sample as itflows through the first pad.
 24. The assay device of claim 20, whereinthe first pad comprises a lysing agent.
 25. The assay device of claim20, wherein the sample comprises blood, serum, plasma, saliva, urine,nasal wash, or a bacterial culture.
 26. The assay device of claim 20,wherein an intermediate pad is interposed between the first pad and thesecond pad, and said intermediate pad has been treated with an immovabledeposit of at least one substance that removes and binds at least onesample component that would otherwise interfere with or obscure theresult of the assay.
 27. The assay device of claim 20, wherein thesample is obtained from a patient.
 28. The assay device of claim 21,wherein the color is formed at a forward flow front of the assay device.29. The assay device of claim 20, wherein the enzyme is selected fromthe group consisting of glucose-6-phosphate deydrogenase (G6PD), alanineaminotransferase, aspartate aminotransferase, lactate dehydrogenase,beta-lactamase, creatine kinase and peroxidase.